多胺氧化酶(PAO)调控光诱导玉米中胚轴伸长的生理机制

Physiological Mechanism Regulating Light-induced Mesocotyl Elongation by Polyamine Oxidase(PAO) in Maize

以长中胚轴玉米自交系PH4CV为材料,在黑暗和光照两种处理条件下研究了玉米中胚轴长度与多胺氧化酶(polyamine oxidase,PAO)活性、H_2O_2含量、过氧化物酶(peroxidase,POD)活性及木质素含量的关系。通过添加5 mmol L^(–1) PAO抑制剂2-羟乙基肼(2-hydroxyethylhydrazine,2-HEH)和5 mmol L^(–1) H_2O_2清除剂N,N’-二甲基硫脲(N,N’-Dimethylthiourea,DMTU)及组织化学染色,研究了影响中胚轴伸长的H_2O_2来源及其积累部位。采用实时荧光定量PCR(q RT-PCR)方法,探究了光对Zm PAO基因表达的影响。结果表明,光照处理显著抑制了玉米中胚轴的伸长,同时显著增加该部位的PAO活性、H_2O_2含量、POD活性及木质素含量。相关性分析表明,玉米中胚轴长度与PAO活性、H_2O_2含量和木质素含量呈极显著负相关,与POD活性呈显著负相关;PAO活性与H_2O_2含量、POD活性和木质素含量均呈正相关。外源PAO抑制剂和H_2O_2清除剂处理试验,玉米中胚轴表皮纵切面细胞长度显微观察及中胚轴H_2O_2组织化学染色表明,PAO氧化分解多胺(polyamines,PAs)产生的H_2O_2参与了中胚轴细胞伸长的生理调控,从而抑制了中胚轴的伸长。表达分析表明,Zm PAO基因在黑暗环境下的表达量相对稳定,光刺激0.5 h后表达量迅速升高,3 h后达到最大值,随后逐渐下降,10 h后趋于稳定。本研究表明,PAO在接受光刺激后活性升高,氧化分解PAs产生H_2O_2,从而诱导POD氧化胞壁的单木质醇并聚合为木质素,使细胞壁硬化,造成细胞伸长受阻。研究结果为进一步探讨PAO活性调控光诱导玉米中胚轴伸长的生理机制和阐明玉米中胚轴对光逆境的响应机理提供了理论依据。

In this study, maize inbred line PH4 CV was cultured under in MS medium under both dark and light conditions to investigate the relationship of length of mesocotyl with activity of polyamine oxidase（PAO）, activity of peroxidase（POD）, contents of H_2O_2 and lignin. In addition, after treated with 5 mmol L^（–1） 2-hydroxyethylhydrazine（2-HEH） and 5 mmol L^（–1） N, N＇-Dimethylthiourea（DMTU）, the origin and accumulation site of H_2O_2 in mesocotyl elongation were assessed by using histochemical staining method and the effect of light on expression of Zm PAO gene was evaluated. The results indicated that the elon-gation of mesocotyl was significantly inhibited while the contents of H_2O_2 and lignin and the activities of POD and PAO were enhanced by light. Correlation analysis showed that the length of mesocotyl was negatively correlated with the PAO activity, H_2O_2 content, POD activity, and lignin content, and the PAO activity was positively correlated with the H_2O_2 content, the POD activity, and the lignin content. The assay with 2-HEH and DMTU, the microgram of epidermal cells, and histochemical localization of H_2O_2 revealed that H_2O_2 participated in mesocotyl elongation, in which PAO catalyzed polyamines（PAs） degradation to produce H_2O_2 resulting in the inhibition of mesocotyl elongation. Results of q RT-PCR revealed that the expression level of Zm PAO was relatively stable in dark and rose rapidly of 0.5 hour after exposing to light, with a maximum value after three hours of light treatment, then declined gradually, and finally showed a steady level after ten hours. This study suggests that the activity of PAO can be promoted by light treatment, and initiate the PAs-mediated induction of H_2O_2, resulting in the oxidation of lignin monomers on cell wall by POD. The produced free radicals are then transformed into lignin in a polyforming process which marked the cell wall hardened and cell elongation inhibited. This study may provide a theoretical basis for understanding the physiological mechanism underlying the PAO-mediated mesocotyl elongation and gain insights into the response of maize mesocotyl to the light stress.